Crystalline forms of 5α-androstane-3β,5,6β-triol and preparation methods therefor

ABSTRACT

The present invention relates to four crystalline forms (crystalline forms A, B, C and D) of 5α-androstane-3β,5,6β-triol (YC-6) and preparation methods therefor. The four crystalline forms have significant difference in their lattice parameters, 2θ values and intensity in X-ray power diffraction, and melting points, etc. The study on its polymorphism is very important for further studying its effect, bioavailability and stability.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of and claims the benefitof priority to U.S. application Ser. No. 14/383,405, filed Sep. 5, 2014,now U.S. Pat. No. 9,688,716, which is a National Stage Entry ofInternational Application No. PCT/CN2012/073854, filed Apr. 11, 2012,which claims priority to Chinese Application No. 201210060611.6, filedMar. 8, 2012. The contents of the above-referenced applications areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to crystalline forms of5α-androstane-3β,5,6β-triol (also known as 5α-androst-3β,5,6β-triol).The present invention also relates to preparation methods for thecrystalline forms of 5α-androstane-3β,5,6β-triol.

BACKGROUND OF THE INVENTION

Polymorphism is common in solid drugs, and drug compounds in differentcrystalline forms have different physical and chemical properties.Polymorphism is one of important factors that have influence on theeffect and quality of solid drugs. Different crystalline forms maydiffer by several times in solubility, and also differ greatly in invivo distribution and metabolism, causing differences inbioavailability. In addition, solid APIs (active pharmaceuticalingredients) in different crystalline forms and preparations thereofshow different stabilities during preparation and storage process, whichwould cause crystalline transformation and thus influence the quality ofthe drugs. Therefore, the polymorphism would finally influence thequality, therapeutic effect and safety of solid drugs.

5α-androstane-3β,5,6β-triol is one of polyhydroxy steroids, and hassignificant neuroprotective effect. The study on its polymorphism isvery important for further studying its efficacy, bioavailability andstability. X-ray single crystal diffractometer, X-ray powderdiffractometer and differential thermal analyzer are main tools forquantitatively determining the specific type of polymorphism, whichprovides more qualitative and quantitative information for the study oncrystalline form of solid drugs.

SUMMARY OF THE INVENTION

An object of the present invention is to provide four crystalline formsof 5α-androstane-3β,5,6β-triol (hereinafter abbreviated as YC-6).

Another object of the present invention is to provide methods forpreparing the four crystalline forms of 5α-androstane-3β,5,6β-triol.

A first crystalline form of 5α-androstane-3β,5,6β-triol (hereinafterabbreviated as crystalline form A of YC-6) is provided by the presentinvention, wherein the crystalline form is a transparent block-shapedcrystal, and belongs to the monoclinic crystal system and space groupP2₁, and wherein the crystalline form is characterized by latticeparameters of a=17.8±0.2 Å, b=7.3±0.2 Å, c=22.1±0.2 Å, α=90.0°,β=103.3±0.5°, γ=90.0°; and characterized by diffraction peaks atdiffraction angle 2θ values of 4.4±0.2, 8.7±0.2, 9.3±0.2, 12.6±0.2,13.0±0.2, 15.0±0.2, 15.6±0.2, 16.6±0.2, 17.3±0.2, 18.5±0.2, 19.6±0.2,21.0±0.2, 21.8±0.2, 24.3±0.2, 27.9±0.2 degrees; and characterized by theendothermic transition temperature of 225±2° C.

A method for preparing the crystalline form A of YC-6 is provided by thepresent invention, comprising: dissolving 5α-androstane-3β,5,6β-triol ina solvent at room temperature or at 50-80° C., with a ratio of the5α-androstane-3β,5,6β-triol to the solvent being 1 g: 10-40 mL; addinganother solvent to dilute; allowing to form a crystalline precipitate.

Preferably, in the above method, the solvent for dissolving is acetone,methanol, ethanol, isopropanol, dioxane or tetrahydrofuran, and thesolvent for diluting is an original solvent or a poor solvent, whereinthe original solvent is acetone, methanol, ethanol, isopropanol ordioxane (excluding tetrahydrofuran) with a dilution rate of 0-5:1, andthe poor solvent is water with a dilution rate of 0-2:1.

A second crystalline form of 5α-androstane-3β,5,6β-triol (hereinafterabbreviated as crystalline form B of YC-6) is provided by the presentinvention, wherein the crystalline form is a transparent needle-shapedcrystal, and belongs to the monoclinic crystal system and space groupP2₁, and wherein the crystalline form is characterized by latticeparameters of a=11.3±0.2 Å, b=7.4±0.2 Å, c=20.5±0.2 Å, α=90.0°,β=95.0±0.5°, γ=90.0°; and characterized by diffraction peaks atdiffraction angle 2θ values of 4.3±0.2, 8.6±0.2, 12.9±0.2, 17.2±0.2,21.6±0.2 degrees; and characterized by the endothermic transitiontemperature of 223±2° C.

A method for preparing the crystalline form B of YC-6 is provided by thepresent invention, comprising: dissolving 5α-androstane-3β,5,6β-triol ina solvent, with a ratio of the 5α-androstane-3β,5,6β-triol to thesolvent being 1 g: 10-120 mL; heating to 50-80° C.; adding anothersolvent to dilute; cooling; allowing to form a crystalline precipitate.

Preferably, in the above method, the solvent for dissolving is acetone,ethyl acetate or ethanol, and the solvent for diluting is an originalsolvent or a poor solvent, wherein the original solvent is acetone,ethyl acetate or ethanol, and the poor solvent is water, hexamethyleneor petroleum ether.

More preferably, in the above method, the dilution rate is 2.5-5:1 whenacetone or ethanol is used as the solvent for dissolving and water isused as the poor solvent for diluting; the dilution rate is 1-5:1 whenacetone or ethanol is used as the solvent for dissolving andhexamethylene or petroleum ether is used as the poor solvent fordiluting; the dilution rate is 0-5:1 when ethyl acetate is used as thesolvent for dissolving and ethyl acetate is used as the original solventfor diluting; and the dilution rate is 0-5:1 when ethyl acetate is usedas the solvent for dissolving and hexamethylene or petroleum ether isused as the poor solvent for diluting.

A third crystalline form of 5α-androstane-3β,5,6β-triol (hereinafterabbreviated as crystalline form C of YC-6) is provided by the presentinvention, wherein the crystalline form is a transparent plate-shapedcrystal, and belongs to the monoclinic crystal system and space groupP2₁, and wherein the crystalline form is characterized by latticeparameters of a=17.1±0.2 Å,b=6.4±0.2 Å, c=34.9±0.2 Å, α=90.0°,β=91.1±0.5°, γ=90.0°; and characterized by diffraction peaks atdiffraction angle 2θ values of 4.2±0.2, 8.5±0.2, 9.0±0.2, 12.5±0.2,14.8±0.2, 15.4±0.2, 16.4±0.2, 16.8±0.2, 17.1±0.2, 18.3±0.2, 19.4±0.2,20.8±0.2, 21.8±0.2, 24.1±0.2 degrees; and characterized by theendothermic transition temperature of 206±2° C.

A method for preparing the crystalline form C of YC-6 is provided by thepresent invention, comprising: dissolving 5α-androstane-3β,5,6β-triol inethanol at room temperature, with a ratio of the5α-androstane-3β,5,6β-triol to the ethanol being 1 g: 10-30 mL; addingethanol in a ratio of 0-5:1 to dilute; allowing to form a crystallineprecipitate at 0-10° C.

A fourth crystalline form of 5α-androstane-3β,5,6β-triol (hereinafterabbreviated as crystalline form D of YC-6) is provided by the presentinvention, wherein the crystalline form is a transparent column-shapedcrystal, and belongs to the orthorhombic crystal system and space groupP2₁2₁2₁, and wherein the crystalline form is characterized by latticeparameters of a=6.3±0.2 Å, b=12.6±0.2 Å, c=26.7±0.2 Å, α=90.0°, β=90°,γ=90.0°; and characterized by diffraction peaks at diffraction angle 2θvalues of 4.0±0.2, 8.1±0.2, 8.5±0.2, 9.4±0.2, 12.5±0.2, 14.0±0.2,14.9±0.2, 15.5±0.2, 16.4±0.2, 17.1±0.2, 18.3±0.2, 19.5±0.2, 20.5±0.2,20.9±0.2, 21.5±0.2 degrees; and characterized by the endothermictransition temperature of 226±2° C.

A method for preparing the crystalline form D of YC-6 is provided by thepresent invention, comprising: dissolving 5α-androstane-3β,5,6β-triol intetrahydrofuran at room temperature, with a ratio of the5α-androstane-3β,5,6β-triol to the tetrahydrofuran being 1 g: 10-30 mL;adding tetrahydrofuran in a ratio of 0-5:1 to dilute; allowing to form acrystalline precipitate.

The four crystalline forms of 5α-androstane-3β,5,6β-triol (i.e.,crystalline forms A, B, C and D of YC-6) provided by the presentinvention have significant differences in their lattice parameters, 28values and intensity in X-ray power diffraction, and melting points,etc. The study on its polymorphism is significant for further studyingof its efficacy, bioavailability and stability.

BRIEF DESCRIPTION OF DRAWING(S)

FIG. 1 shows the X-ray single crystal diffraction diagrams ofcrystalline form A of YC-6.

FIG. 2 shows the X-ray powder diffraction diagrams of crystalline form Aof YC-6.

FIG. 3 shows the differential thermal analysis diagrams of crystallineform A of YC-6.

FIG. 4 shows the X-ray single crystal diffraction diagrams ofcrystalline form B of YC-6.

FIG. 5 shows the X-ray powder diffraction diagrams of crystalline form Bof YC-6.

FIG. 6 shows the differential thermal analysis diagrams of crystallineform B of YC-6.

FIG. 7 shows the X-ray single crystal diffraction diagrams ofcrystalline form C of YC-6.

FIG. 8 shows the X-ray powder diffraction diagrams of crystalline form Cof YC-6.

FIG. 9 shows the differential thermal analysis diagrams of crystallineform C of YC-6.

FIG. 10 shows the X-ray single crystal diffraction diagrams ofcrystalline form D of YC-6.

FIG. 11 shows the X-ray powder diffraction diagrams of crystalline formD of YC-6.

FIG. 12 shows the differential thermal analysis diagrams of crystallineform D of YC-6.

DETAILED DESCRIPTION OF THE INVENTION

Physical Characterization

X-ray single crystal diffraction diagrams for different crystallineforms of YC-6, which were obtained by the examples, were obtained usingXcalibur Nova biomacromolecule X-Ray single crystal diffractometer(Agilent Technologies (China) Co., Ltd), under the following settings:fixed target of copper; output power: 50 W; two-dimensional surfacedetecting system: 165 mmCCD; resolution: ≤0.005 degree; coolingnitrogen: −180+ approx. 25° C.; control accuracy: ≤0.5° C.; testtemperature: 150 k.

X-ray powder diffraction diagrams for different crystalline forms ofYC-6, which were obtained by the examples, were obtained usingD/Max-IIIA X-ray powder diffractometer (Rigaku, Japan), under thefollowing settings: fixed target of copper; power: 3 kW; detectingangle: 1-50°; sensitivity: 3-5%; accuracy of the detecting angle:±0.002.

Differential scanning for different crystalline forms of YC-6, whichwere obtained by the examples, were performed using STA409PC thermalanalyzer (Netzsch, Germany), under the following settings: crucible:alumina crucible; carrier gas: N₂; temperature: 20-400° C., 10.0 K/min,400° C. maintained by 10 min.

Analysis Parameters of the Single Crystal Diffraction, PowderDiffraction and DSC for the Four Crystalline Forms of YC-6

(1) The crystal structure information of crystalline form A of YC-6obtained by X-ray single crystal diffraction is: the crystalline formbelongs to the monoclinic crystal system and space group P2₁, withlattice parameters of a=17.76±0.08 Å, b=7.30±0.08 Å, c=22.05±0.08 Å,α=90.0°, β=103.23±0.5°, γ=90.0°, V=2775.36(5) A³.

The crystalline form A of YC-6 showed diffraction peaks at diffractionangle 2θ values of 4.4±0.1, 8.7±0.1, 9.3±0.1, 12.6±0.1, 13.0±0.1,15.0±0.1, 15.6±0.1, 16.6±0.1, 17.3±0.1, 18.5±0.1, 19.6±0.1, 21.0±0.1,21.8±0.1, 24.3±0.1, 27.9±0.1 degrees, with the X-ray powder diffractiondiagrams showed in FIG. 2.

The differential scanning calorimetry (DSC) diagrams of crystalline formA of YC-6 is showed in FIG. 3, with the endothermic transitiontemperature of 225±2° C.

(2) The crystal structure information of crystalline form B of YC-6obtained by X-ray single crystal diffraction is: the crystalline formbelongs to the monoclinic crystal system and space group P2₁, withlattice parameters of a=11.27±0.08 Å, b=7.40±0.08 Å, c=20.45±0.08 Å,α=90.0°, β=94.94±0.5°, γ=90.0°, V=1699.24(3) A³.

The crystalline form B of YC-6 showed diffraction peaks at diffractionangle 2θ values of 4.3±0.1, 8.6±0.1, 12.9±0.1, 17.2±0.1, 21.6±0.1degrees, with the X-ray powder diffraction diagrams showed in FIG. 5.

The differential scanning calorimetry (DSC) diagrams of crystalline formB of YC-6 is showed in FIG. 6, with the endothermic transitiontemperature of 223±2° C.

(3) The crystal structure information of crystalline form C of YC-6obtained by X-ray single crystal diffraction is: the crystalline formbelongs to the monoclinic crystal system and space group P2₁, withlattice parameters of a=17.14±0.08 Å, b=6.40±0.08 Å, c=34.89±0.08 Å,α=90.0°, β=91.05±0.5°, γ=90.0°, V=3827.48(9) A³.

The crystalline form C of YC-6 showed diffraction peaks at diffractionangle 2θ values of 4.2±0.1, 8.5±0.1, 9.0±0.1, 12.5±0.1, 14.8±0.1,15.4±0.1, 16.4±0.1, 16.8±0.2, 17.1±0.1, 18.3±0.1, 19.4±0.1, 20.8±0.1,21.8±0.1, 24.1±0.1 degrees, with the X-ray powder diffraction diagramsshowed in FIG. 8.

The differential scanning calorimetry (DSC) diagrams of crystalline formC of YC-6 is showed in FIG. 9, with the endothermic transitiontemperature of 206±2° C.

(4) The crystal structure information of crystalline form D of YC-6obtained by X-ray single crystal diffraction is: the crystalline formbelongs to the orthorhombic crystal system and space group P2₁2₁2₁, withlattice parameters of a=6.28±0.08 Å, b=12.56±0.08 Å, c=26.68±0.08 Å,α=90.0°, β=90.0°, γ=90.0°, V=2103.09(7) A³.

The crystalline form D of YC-6 showed diffraction peaks at diffractionangle 2θ values of 4.0±0.1, 8.1±0.1, 8.5±0.1, 9.4±0.1, 12.5±0.1,14.0±0.1, 14.9±0.1, 15.5±0.1, 16.4±0.1, 17.1±0.1, 18.3±0.1, 19.5±0.1,20.5±0.1, 20.9±0.1, 21.5-0.1 degrees, with the X-ray powder diffractiondiagrams showed in FIG. 11.

The differential scanning calorimetry (DSC) diagrams of crystalline formD of YC-6 is showed in FIG. 12, with the endothermic transitiontemperature of 226±2° C.

Example 1

Preparation of crystalline form A of YC-6: 0.5 g of YC-6 was dissolvedin 8 mL of acetone (50-60° C.), which was added by the same amount ofacetone to dilute, and then allowed to form a crystalline precipitate.The single crystal obtained thereby was directly subjected to X-raysingle crystal diffraction. Then the crystal was filtrated by suctionand was air-dried at 60° C. to a constant weight, which was subjected toX-ray powder diffraction and differential scanning calorimetry.

Example 2

Preparation of crystalline form A of YC-6: 0.5 g of YC-6 was dissolvedin 10 mL of acetone at room temperature, which was added by the sameamount of acetone to dilute, and then allowed to form a crystallineprecipitate. The single crystal obtained thereby was directly subjectedto X-ray single crystal diffraction. Then the crystal was filtrated bysuction and was air-dried at 60° C. to a constant weight, which wassubjected to X-ray powder diffraction and differential scanningcalorimetry.

Example 3

Preparation of crystalline form A of YC-6: 0.5 g of YC-6 was dissolvedin 7 mL of ethanol at room temperature, which was added by the sameamount of ethanol to dilute, and then allowed to form a crystallineprecipitate. The single crystal obtained thereby was directly subjectedto X-ray single crystal diffraction. Then the crystal was filtrated bysuction and was air-dried at 60° C. to a constant weight, which wassubjected to X-ray powder diffraction and differential scanningcalorimetry.

Example 4

Preparation of crystalline form A of YC-6: 0.5 g of YC-6 was dissolvedin 12 mL of acetone at room temperature, which was added by water (ahalf amount of that of the acetone) to dilute, and then allowed to forma crystalline precipitate. The single crystal obtained thereby wasdirectly subjected to X-ray single crystal diffraction. Then the crystalwas filtrated by suction and was air-dried at 60° C. to a constantweight, which was subjected to X-ray powder diffraction and differentialscanning calorimetry.

Example 5

Preparation of crystalline form A of YC-6: 0.5 g of YC-6 was dissolvedin 10 mL of ethanol at room temperature, which was added by water (ahalf amount of that of the ethanol) to dilute, and then allowed to forma crystalline precipitate. The single crystal obtained thereby wasdirectly subjected to X-ray single crystal diffraction. Then the crystalwas filtrated by suction and was air-dried at 60° C. to a constantweight, which was subjected to X-ray powder diffraction and differentialscanning calorimetry.

The tests showed that the crystals obtained in examples 1-5 share thesame lattice parameters in X-ray single crystal diffraction, and thatthe crystals obtained thereby are all crystalline form A of YC-6.

Example 6

Preparation of crystalline form B of YC-6: 0.5 g of YC-6 was dissolvedin 30 mL of ethyl acetate, which was then heated to 70-80° C. Afterthat, the solution was added by 30 mL of ethyl acetate to dilute, andthen cooled and allowed to form a crystalline precipitate. The singlecrystal obtained thereby was directly subjected to X-ray single crystaldiffraction. Then the crystal was filtrated by suction and was air-driedat 70° C. to a constant weight, which was subjected to X-ray powderdiffraction and differential scanning calorimetry.

Example 7

Preparation of crystalline form B of YC-6: 0.5 g of YC-6 was dissolvedin 30 mL of ethyl acetate, which was then heated to 70-80° C. Afterthat, the solution was added by 30 mL of hexamethylene to dilute, andthen cooled and allowed to form a crystalline precipitate. The singlecrystal obtained thereby was directly subjected to X-ray single crystaldiffraction. Then the crystal was filtrated by suction and was air-driedat 70° C. to a constant weight, which was subjected to X-ray powderdiffraction and differential scanning calorimetry.

Example 8

Preparation of crystalline form B of YC-6: 0.5 g of YC-6 was dissolvedin 8 mL of acetone, which was then heated to 50-60° C. After that, thesolution was added by 24 mL of water to dilute, and then cooled andallowed to form a crystalline precipitate. The single crystal obtainedthereby was directly subjected to X-ray single crystal diffraction. Thenthe crystal was filtrated by suction and was air-dried at 70° C. to aconstant weight, which was subjected to X-ray powder diffraction anddifferential scanning calorimetry.

Example 9

Preparation of crystalline form B of YC-6: 0.5 g of YC-6 was dissolvedin 12 mL of acetone, which was then heated to 50-60° C. After that, thesolution was added by 36 mL of hexamethylene to dilute, and then cooledand allowed to form a crystalline precipitate. The single crystalobtained thereby was directly subjected to X-ray single crystaldiffraction. Then the crystal was filtrated by suction and was air-driedat 70° C. to a constant weight, which was subjected to X-ray powderdiffraction and differential scanning calorimetry.

The tests showed that the crystals obtained in examples 6-9 share thesame lattice parameters in X-ray single crystal diffraction, and thatthe crystals obtained thereby are all crystalline form B of YC-6.

Example 10

Preparation of crystalline form C of YC-6: 0.5 g of YC-6 was dissolvedin 12 mL of ethanol at room temperature, which was added by the sameamount of ethanol to dilute, and then allowed to form a crystallineprecipitate at 10° C. The single crystal obtained thereby was directlysubjected to X-ray single crystal diffraction. Then the crystal wasfiltrated by suction and was air-dried at 70° C. to a constant weight,which was subjected to X-ray powder diffraction and differentialscanning calorimetry.

Example 11

Preparation of crystalline form C of YC-6: 0.5 g of YC-6 was dissolvedin 15 mL of ethanol at room temperature, which was added by the sameamount of ethanol to dilute, and then allowed to form a crystallineprecipitate at 10° C. The single crystal obtained thereby was directlysubjected to X-ray single crystal diffraction. Then the crystal wasfiltrated by suction and was air-dried at 70° C. to a constant weight,which was subjected to X-ray powder diffraction and differentialscanning calorimetry.

Example 12

Preparation of crystalline form C of YC-6: 0.5 g of YC-6 was dissolvedin 15 mL of ethanol at room temperature, which was added by twice amountof ethanol to dilute, and then allowed to form a crystalline precipitateat 10° C. The single crystal obtained thereby was directly subjected toX-ray single crystal diffraction. Then the crystal was filtrated bysuction and was air-dried at 70° C. to a constant weight, which wassubjected to X-ray powder diffraction and differential scanningcalorimetry.

The tests showed that the crystals obtained in examples 10-12 share thesame lattice parameters in X-ray single crystal diffraction, and thatthe crystals obtained thereby are all crystalline form C of YC-6.

Example 13

Preparation of crystalline form D of YC-6: 0.5 g of YC-6 was dissolvedin 10 mL of tetrahydrofuran at room temperature, which was added by thesame amount of tetrahydrofuran to dilute, and then allowed to form acrystalline precipitate. The single crystal obtained thereby wasdirectly subjected to X-ray single crystal diffraction. Then the crystalwas filtrated by suction and was air-dried at 70° C. to a constantweight, which was subjected to X-ray powder diffraction and differentialscanning calorimetry.

Example 14

Preparation of crystalline form D of YC-6: 0.5 g of YC-6 was dissolvedin 10 mL of tetrahydrofuran at room temperature, which was added bytwice amount of tetrahydrofuran to dilute, and then allowed to form acrystalline precipitate. The single crystal obtained thereby wasdirectly subjected to X-ray single crystal diffraction. Then the crystalwas filtrated by suction and was air-dried at 70° C. to a constantweight, which was subjected to X-ray powder diffraction and differentialscanning calorimetry.

Example 15

Preparation of crystalline form D of YC-6: 0.5 g of YC-6 was dissolvedin 15 mL of tetrahydrofuran at room temperature, which was added by thesame amount of tetrahydrofuran to dilute, and then allowed to form acrystalline precipitate. The single crystal obtained thereby wasdirectly subjected to X-ray single crystal diffraction. Then the crystalwas filtrated by suction and was air-dried at 70° C. to a constantweight, which was subjected to X-ray powder diffraction and differentialscanning calorimetry.

The tests showed that the crystals obtained in examples 13-15 share thesame lattice parameters in X-ray single crystal diffraction, and thatthe crystals obtained thereby are all crystalline form D of YC-6.

The above examples are merely provided for description of the presentinvention, and are not intended to limit the scope of the presentinvention. The objects of the present invention can be achieved byskilled persons in the art in accordance with the disclosure of thepresent invention and the parameter ranges involved.

What is claimed is:
 1. A crystalline form of5α-androstane-3β,5,6β-triol, wherein the crystalline form is atransparent plate-shaped crystal, and belongs to monoclinic crystalsystem and space group P2₁, and wherein the crystalline form ischaracterized by lattice parameters of a=17.1±0.2 Å, b=6.4±0.2 Å,c=34.9±0.2 Å, α=90.0°, β=91.1±0.5°, γ=90.0°; and characterized bydiffraction peaks at diffraction angle 2θ values of 4.2±0.2, 8.5±0.2,9.0±0.2, 12.5±0.2, 14.8±0.2, 15.4±0.2, 16.4±0.2, 16.8±0.2, 17.1±0.2,18.3±0.2, 19.4±0.2, 20.8±0.2, 21.8±0.2, 24.1±0.2 degrees; andcharacterized by an endothermic transition temperature of 206±2° C.
 2. Amethod for preparing the crystalline form of claim 1, comprising:dissolving 5α-androstane-3β,5,6β-triol in ethanol at room temperature,with a ratio of the 5α-androstane-3β,5,6β-triol to the ethanol being 1g:10-30 mL; and allowing to form a crystalline precipitate at 0-10° C.3. The method of claim 2, wherein the method comprises, after thedissolving, adding ethanol to dilute.
 4. The method of claim 3, whereinthe dilution is performed at a ratio less than 5:1.